Novel Substituted Purine Isosteres: Synthesis, Structure-Activity Relationships and Cytotoxic Activity Evaluation

A number of pyrrolo[2,3-c]pyridines, pyrrolo[3,2-d]pyrimidines and pyrazolo[4,3-d]pyrimidines were designed and synthesized as antiproliferative agents. The target compounds possessed selected substituents in analogous positions on the central scaffold that allowed the extraction of interesting SARs. The cytotoxic activity of the new derivatives was evaluated against prostatic (PC-3) and colon (HCT116) cell lines, and the most potent analogues showed IC50 values in the nM to low µM range, while they were found to be non-toxic against normal human fibroblasts (WI-38). Flow cytometric analysis of DNA content revealed that the most promising derivative 14b caused a statistically significant accumulation of PC-3 cells at G2/M phase and induced apoptosis in PC-3 cells.

reduction of the oxime group to result in the aminopyrazole-carboxylates 32a [36] and 32b, in moderate yields (40%). The reaction of the carboxylates 32a,b with formamidine gave the pyrazolopyrimidinones 33a [37] and 33b, which-through the corresponding chlorides 34a,b and the displacement of the chlorine by suitable amines-resulted in the target pyrazolopyrimidines 35a-f in good overall yields (over 40% for three steps). All new compounds were unambiguously identified using NMR and mass spectrometry data.

Biological Evaluation
The cytotoxic activity of the new compounds was tested against two cancer cell lines, prostatic (PC-3) and colon (HCT116) cell lines (Table 1), while the most potent derivatives were further tested against normal human fibroblasts (WI-38) ( Table 2).
Concerning the N-(p-methoxybenzyl) substituted pyrrolopyridines 10a-d and 13a,b, only a few of them possessed moderate antiproliferative activity, namely the 7-phenylamino derivative 10a, together with both the 7-(N-methylpiperazine) substituted derivatives 13a,b, with IC50 values in the range of 2.4-9.3 µM. On the other hand, the deprotection of the above-mentioned compounds provided derivatives 14a-f, which were endowed with very potent cytotoxic properties, with IC50 values in the nM to low µM range against both cell lines tested. The most potent compound of this group is the 3-(3-fluorophenyl) analogue 14b, possessing IC50 values of 55nM and 90 nM against the prostate and colon cell lines, respectively.

Biological Evaluation
The cytotoxic activity of the new compounds was tested against two cancer cell lines, prostatic (PC-3) and colon (HCT116) cell lines (Table 1), while the most potent derivatives were further tested against normal human fibroblasts (WI-38) ( Table 2).
Concerning the N-(p-methoxybenzyl) substituted pyrrolopyridines 10a-d and 13a,b, only a few of them possessed moderate antiproliferative activity, namely the 7-phenylamino derivative 10a, together with both the 7-(N-methylpiperazine) substituted derivatives 13a,b, with IC 50 values in the range of 2.4-9.3 µM. On the other hand, the deprotection of the above-mentioned compounds provided derivatives 14a-f, which were endowed with very potent cytotoxic properties, with IC 50 values in the nM to low µM range against both cell lines tested. The most potent compound of this group is the 3-(3-fluorophenyl) analogue 14b, possessing IC 50 values of 55nM and 90 nM against the prostate and colon cell lines, respectively.
In the pyrrolopyrimidine series, the N-(p-methoxybenzyl) substituted analogues 44ad and 25a,b are, in analogy to the previous series, devoid of activity, with the sole exception of 24c, which appeared to be cytotoxic against PC-3 cells (IC 50 : 2.25 µM). Concerning the corresponding deprotected analogues, the 7-(3-fluorophenyl)-4-phenylamino derivative 26b is the most potent compound of this group, followed by the corresponding 7-phenylsubstituted compound 26a. The 4-(3,4,5-trimethoxyphenyl)amino-substituted derivatives 26c,d had moderate activity, while a complete loss of activity was observed in the corresponding 4-(N-methylpiperazine)-substituted analogues.
Finally, in the case of pyrazolopyrimidines, the 3-(3-fluorophenyl)-7-phenylamino derivative 35b possessed very interesting cytotoxicity, whereas among the remaining compounds, only 35a and 35c showed moderate cytotoxicity, and again the N-methylpiperazine substituted analogues were inactive. Physicochemical characteristics of the thirty compounds tested were calculated using SwissADME platform [38] and are summarized in Table S1. They were further entered as inputs along with the IC 50 s in the SIMCA software [39]. A multivariate analysis showed that there is a very weak correlation (R2 < 0.25) between activity and molar refractivity, while there was no correlation between activity and logP. Table 1. Accumulative results of the antiproliferative activities of all compounds tested.

Compd
Physicochemical characteristics of the thirty compounds tested were calculated using SwissADME platform [38] and are summarized in Table S1. They were further entered as inputs along with the IC50s in the SIMCA software [39]. A multivariate analysis showed that there is a very weak correlation (R2 < 0.25) between activity and molar refractivity, while there was no correlation between activity and logP.
As a general remark, it could be stated that the cytotoxic activity is enhanced in the absence of substituents on the pyrrole or pyrazole nitrogen of the studied compounds and that the pyrrolopyridines possess the most interesting profile, since all six derivatives (14a-f) are potent against both cell lines tested. As already mentioned, 14b is the most active derivative, and at the same time, the analogously substituted pyrrolopyrimidine 26b and pyrazolopyrimidine 35b are also endowed with strong cytotoxic properties. It is of interest to notice that the prostatic cell line appears the most sensitive to the compounds.  As a general remark, it could be stated that the cytotoxic activity is enhanced in the absence of substituents on the pyrrole or pyrazole nitrogen of the studied compounds and that the pyrrolopyridines possess the most interesting profile, since all six derivatives (14a-f) are potent against both cell lines tested. As already mentioned, 14b is the most active derivative, and at the same time, the analogously substituted pyrrolopyrimidine 26b and pyrazolopyrimidine 35b are also endowed with strong cytotoxic properties. It is of interest to notice that the prostatic cell line appears the most sensitive to the compounds.
The cytotoxicity of the most potent derivatives, i.e., the majority of the pyrrolopyridines 14, together with 26b and 35b, was then examined towards normal human fibroblasts (WI-38); the results are presented in Figure 1 and Table 2.
Among them, 14a and 14c-e appear to be rather cytotoxic to normal cells. By contrast, it is important to note that the 7-phenylamino substituted compound 14b that bears a 3-(3-fluorophenyl)-group, while being highly cytotoxic against both cancer cell lines, appears practically non-toxic against the normal cell line, presenting a selectivity index higher than 200. The derivatives 26b and 35b that possess the same substitution pattern in the central scaffold retain potent activity against the cancer cell lines and appear noncytotoxic to the normal cell line as well, albeit to a lesser extent than 14b.  Among them, 14a and 14c-e appear to be rather cytotoxic to normal cells. By contrast, it is important to note that the 7-phenylamino substituted compound 14b that bears a 3-(3- fluorophenyl)-group, while being highly cytotoxic against both cancer cell lines, appears practically non-toxic against the normal cell line, presenting a selectivity index higher than 200. The derivatives 26b and 35b that possess the same substitution pattern in the central scaffold retain potent activity against the cancer cell lines and appear non-cytotoxic to the normal cell line as well, albeit to a lesser extent than 14b.
Cell-cycle perturbations induced after the incubation of exponentially growing PC-3 cells with compounds 14b, 26b and 35b for 72 h are given in Figure 2 and Table 3. Compound 14b caused a statistically significant accumulation of PC-3 cells at G 2 /M phase, significantly reducing, in parallel, the percentage of cells at G 0 /G 1 phase (the accumulation of cells at S phase was marginally non-significant).
1 Figure 2. Flow cytometric graphs of DNA content. Indicative DNA content histograms of PC-3 cells treated for 72 h with 14b, 26b and 35b, in comparison to control cells (vehicle). Quantification of cell cycle phase distribution was based on staining by the DNA binding dye propidium iodide. Doxorubicin, which was used as a positive control at a concentration of 25 nM, showed the expected G2/M phase blockade (58%) and the induction of apoptosis (28%), as previously reported [40]. Furthermore, 14b, 26b and 35b induced apoptosis in PC-3 cells after 72 h of treatment, as estimated by AnnexinV-7AAD staining. In particular, compound 14b induced the appearance of 26.5% (±0.47) apoptotic nuclei, 26b induced the appearance of 17.7% (±1.8) apoptotic nuclei and 35b induced the appearance of 11.5% (±0. 19) apoptotic nuclei, compared to 3.5% (±0.05) induced by the vehicle (Figure 3). These data give an indication that the mechanism of action of compound 14b may include blocking of the cell cycle at the G 2 /M phase and inducing apoptosis.

General Information
The reagents and solvents were purchased from Sigma-Aldrich Chemical Co. (Darmstadt, Germany) or Fluorochem (Derbyshire's Peak District, UK). Reagents were used without further purification. Concerning the dry solvents, methanol and dimethylformamide were dried over 3A and 4A molecular sieves, respectively; toluene was pre-dried using CaH 2 and then placed over sodium. Hydrazine is considered a suspect carcinogen in humans, may cause serious damage and should be handled carefully. Melting points were determined on a Büchi apparatus (Flawil, Switzerland) and are uncorrected. 1 HNMR spectra and 2D spectra were recorded on a Bruker Avance III 600 or a Bruker Avance DRX 400 instrument (Bruker BioSpin, Baden-Württemberg, Germany), whereas 13 CNMR spectra were recorded on a Bruker Avance III 600 spectrometer in deuterated solvents and were referenced to TMS (δ scale). The signals of 1 H and 13 C spectra were unambiguously assigned by using 2D NMR techniques: 1 H 1 H COSY, NOESY, HMQC and HMBC. Mass spectra were recorded with a LTQ Orbitrap Discovery instrument (Thermo Scientific, Dreieich, Germany), possessing an Ionmax ionization source. Flash chromatography was performed on Merck silica gel 60 (0.040-0.063 mm). The purity of all the target compounds that underwent biological evaluation was >95%, as ascertained by elemental analysis. Elemental analyses were undertaken using a PerkinElmer PE 240C elemental analyzer (Norwalk, CT, U.S.) and the measured values for C, H and N were within ± 0.4% of the theoretical values. Analytical thin layer chromatography (TLC) was carried out on precoated (0.25 mm) Merck silica gel F-254 plates (Merck KGa, Darmstadt, Germany). The controlled injection of solutions was performed with the Bioblock Scientific device (Illkirch, France). (7): This compound is mentioned in a patent [41]; here, we provide the methodology and identification data. To a solution of 6 (1.40 g, 9.15 mmol) in anhydrous methanol (50 mL), N-iodosuccinimide (2.80 g, 12.4 mmol) was added, and the mixture was stirred at rt for 1 h. Then, the organic solvent was evaporated under vacuo, and the residue was extracted with ethyl acetate. The organic layer was washed with a 10% sodium thiosulfate aqueous solution, dried (Na 2 SO 4 ) and evaporated to dryness to result in 7 (2. 1-(4-Methoxybenzyl)-7-(4-methylpiperazin-1-yl)-3-phenyl-1H-pyrrolo[2,3-c]pyridine-6-oxide (12a): To a solution of 11a (120 mg, 0.33 mmol) in absolute ethanol (5 mL), 1methylpiperazine (0.20 mL, 2.00 mmol) was added, and the solution was heated in an autoclave at 120 • C for 24 h. The mixture was extracted with ethyl acetate, the organic phase was dried (Na 2 SO 4 ) and concentrated to dryness and the residue was purified by silica gel column chromatography (dichloromethane/methanol: 8/2) to give 12a (40 mg, 50%), m.p. 127-128 • C (EtOAc/n-pentane). 1 H NMR (600 MHz, CD 3 OD) 8.00 (d, J = 6.  [3,2-d] pyrimidine (25b): This compound was synthesized using a procedure analogous to that of 25a, starting from 23b. Purification was carried out by silica gel column chromatography (dichloromethane/methanol: 9/1). Yield: 97%, m.p. 151-152 • C (CH 2 Cl 2 /n-pentane). 1 H NMR (600 MHz, Acetone-d 6 13  This compound was synthesized using a procedure analogous to that of 34a, starting from 33b. Purification was carried out by silica gel column chromatography (cyclohexane/ethyl acetate: 7/3). Yield: dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma M-5655) was added at a final concentration of 0.5 mg/mL directly to each well for 4 h at 37 • C. The medium was aspirated, and the blue MTT formazan precipitate was dissolved in dimethyl sulfoxide (DMSO). Absorbance was determined in a Powerwave microplate spectrophotometer (Biotek Instruments, Inc.) at 540 nm. Viable cell numbers were determined by tetrazolium conversion to its formazan dye. The IC 50 was calculated by Microsoft Excel equation and confirmed by GraphPad Prism (7.0). Each experiment was performed in triplicate and mean values ± SD are reported.

DNA Staining and Flow Cytometric Analysis
Exponentially growing PC-3 cells were treated with the IC 50 values of the compounds 14b, 26b and 35b or the corresponding DMSO concentration (vehicle) for 72 h. For cell cycle analysis, cell culture supernatants and attached cells were collected, centrifuged, washed in PBS, fixed in 50% ethanol and stained with an RNase-containing propidium iodide solution (50 µg/mL) (all reagents from Sigma). For cell apoptosis assay, cells were harvested and stained with Annexin V binding buffer, Annexin V-FITC and 7-AAD (Annexin V-FITC Apoptosis Detection Kit, BD Systems). DNA content was analyzed on a BD Accuri C6 Flow Cytometer using the BD CSampler software (BD Biosciences, USA). Non-apoptotic events were used to calculate the percentage of cells distributed in each phase. A p value < 0.05 was considered to be statistically significant (Student's t-test).

Conclusions
In conclusion, we have designed and synthesized a number of new substituted pyrrolo [2,3-c]pyridines, pyrrolo [3,2-d]pyrimidines and pyrazolo [4,3-d]pyrimidines and have evaluated their antiproliferative activity against two cancer cell lines. We preserved an analogous substitution pattern around the fused ring-system of all three scaffolds in order to assist the extraction of SARs. We have identified a number of derivatives with potent cytotoxic properties-more precisely, the pyrrolopyridines 14a-f, the pyrrolopyrimidines 26a,b and the pyrazolopyrimidines 35a,b, all of which possess IC 50 values in the nM to low µM level against both cell lines tested. We assume that in all cases the simultaneous presence of a 3-fluorophenyl moiety and a phenylamino substituent at analogous positions resulted in highly active compounds which induce apoptosis in PC-3 cells and, importantly, are devoid of toxicity against normal cells. These interesting observations have triggered the continuation of research efforts in our laboratory for the investigation of the exact mechanism of action of this promising class of compounds.
Supplementary Materials: The following supporting information can be downloaded online: Figures S1-S55: 1 H-NMR and 13 C NMR spectra of new compounds, HRMS spectra of active compounds; Table S1: Calculated physicochemical characteristics of the tested compounds using SwissADME.